PROJECT SUMMARY ? TR&D1 In this project, we focus on development of functional genomics technologies for use in Drosophila cells and/or in vivo. Studies in cells provide a platform for large-scale gene function discovery using loss-of-function (e.g. CRIPSR knockout and CRISPR interference) and gain-of-function (e.g. CRISPR activation) approaches, and serves as an efficient platform to test technologies prior to their applications in vivo. Our plans balance relatively low-risk refinement of Drosophila pooled screen approaches with higher-risk but potentially highly impactful development of new CRISPR/Cas libraries and applications, e.g. using variant Cas9 forms with alternative protospacer-adjacent motifs, CRISPR inhibition (CRISPRi), and/or CRISPR-based targeting of RNA. A set of collaborators with interest in using screens and in vivo technologies to study cancer, rare diseases, cell biology, and other topics will help test and improve the technologies, and for screens in particular, the accumulation of data from multiple projects will help improve reagent design, data analysis, off- target prediction, etc. Altogether, this project will establish CRISPR pooled-format screens as a robust platform for analysis of conserved gene function, as well as result in new and improved cell and in vivo approaches, contributing to effective and efficient use of Drosophila to study health and disease.